1. Field of the Invention
The present invention relates to novel sulfofucosylacylglycerol derivatives. The novel sulfofucosylacylglycerol derivatives of the present invention are useful as medicaments, more specifically, a DNA polymerase inhibitor and an anticancer agent.
2. Description of the Related Art
Sulfur-containing glycolipids contained in natural products derived from, e.g., algae and higher plants are known to have physiological activities.
For example, in a document of Ohta et al. (Chemical and Pharmaceutical Bulletin, 46(4), (1998)), it is described that a specific sulfoquinovosyldiacylglycerol derivative derived from red algae, Gigartina tenella, exhibits not only inhibitory activities against DNA polymerases xcex1 and xcex2 of higher organisms but also an inhibitory activity against HIV-derived reverse-transcriptase.
Furthermore, in a document of Mizushina et al. (Biochemical Pharmacology 55, 537-541 (1998)), it is described that specific sulfoquinovosyldiacylglycerol derivatives derived from a pteridophyte exhibits inhibitory activities against a calf DNA polymerase a and a rat DNA polymerase xcex2, but does not have any influence on the inhibitory activity against HIV-derived reverse-transcriptase.
On the other hand, in a document of Sahara et al. (British Journal of Cancer, 75(3), 324-332 (1997)), it is described that a fraction of sulfoquinovosylmonoacylglycerols obtained from sea urchin intestine exhibits anticancer activities in-vivo and in-vitro.
However, sulfur-containing glycolipids disclosed in Ohta et al., Mizushina et al., and Sahara et al., are sulfoquinovosylacylglycerol derivatives having an xcex1-quinovose (i.e., 6-deoxy-xcex1-glucose) as a sugar component thereof. A sulfur-containing glycolipids having a fucose (i.e., 6-deoxygalactose) as a sugar component has not yet been known.
Furthermore, National Patent Publication No. 5-501105 describes that a sulfoquinovosyldiacylglycerol derivative has an anti-virus activity. More specifically, it discloses that the derivative has an anti-HIV (human immunodeficiency virus) activity, however it does not disclose that the derivative has inhibitory activities against DNA polymerase and anticancer activities.
An object of the present invention is to provide a novel sulfofucosylacylglycerol derivative having a fucose as a sugar component and its use as a medicament.
The present invention provides compounds represented by the following General Formula (1): 
wherein R101 represents an acyl residue of a higher fatty acid, and R102 represents a hydrogen atom or an acyl residue of a higher fatty acid.
Furthermore, the present invention also provides medicaments containing, as an active ingredient, at least one compound selected from the group consisting of the compounds represented by General Formula (1) and pharmaceutically acceptable salts thereof.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
In the specification, the term xe2x80x9ccarbon atomsxe2x80x9d of a protecting group refers to the number of carbon atoms assuming that the protecting group is unsubstituted. To be more specific, when the group represented by R1 is a substituted alkyl group, its number of carbon atoms is that of the alkyl group itself, and the number of carbon atoms of the substituent on the alkyl group is not counted. The same conditions are applicable to the case where the protecting group is other than the alkyl group.
First, the sulfofucosylacylglycerol derivative (hereinafter, also referred to as xe2x80x9csulfofucosylacylglycerol derivative of the present inventionxe2x80x9d) represented by General Formula (1) of the present invention will be more specifically explained.
The sulfofucosylacylglycerol derivative of the present invention is represented by following General Formula (1): 
wherein R101 represents an acyl residue of a higher fatty acid, and R102 represents a hydrogen atom or an acyl residue of a higher fatty acid.
In General Formula (1), RIO represents an acyl residue of a higher fatty acid. The fatty acids providing the acyl residues represented by R101 includes straight-chain or branched-chain, saturated or unsaturated higher fatty acids.
When the sulfofucosylacylglycerol derivative of the present invention is used as a medicament, R101 is preferably an acyl residue of a straight-chain saturated higher fatty acid in view of its anticancer activity, in particular, against a solid tumor, for example, gastric cancer and colon cancer, and more preferably a group represented by CH3(CH2)nCOxe2x80x94 (wherein n is an integer of 12-24, preferably an even number of 12-24). The present inventors predict that sulfofucosylacylglycerol derivatives, where R101 of General Formula (1) of the invention is represented by CH3(CH2)nCOxe2x80x94 (n greater than 24), may also have an anticancer activity. However, the sulfofucosylacylglycerol derivatives having such long-chain acyl residues are not used in practice in view of manufacturing cost and the like.
In General Formula (1) mentioned above, R102 represents a hydrogen atom or an acyl residue of a higher fatty acid. The fatty acids providing the acyl residues include straight-chain or branched-chain, saturated or unsaturated higher fatty acids, and more specifically, include the same fatty acids as those mentioned above for R101.
When the sulfofucosylacylglycerol derivatives of the present invention are used as a medicament, R102 is preferably a hydrogen atom in view of their anticancer activities, in particular, against solid tumor, for example, gastric cancer and colon cancer.
In General formula (1), the sugar skeleton of the sulfofucoside may be either a boat or chair configuration. However, the chair configuration is preferable in view of stability. Furthermore, the bonding between sulfofucose and glycerol is either an xcex1- or xcex2-bonding. Furthermore, the absolute configuration of the carbon (asymmetric carbon) at the 2-position of the glycerol moiety may be either the S- or R-configuration.
Now, a method of preparing the sulfofucosylacylglycerol derivatives of the present invention will be explained below.
The sulfofucosylacylglycerol derivatives of the present invention can be prepared via (Step A) to (Step J) in accordance with the reaction procedure shown in Scheme 1 below: 
(Step A) The hydroxyl group bonded to the C1 carbon of the D-galactose is converted into a 2-propenyl group. (Step B) The hydroxyl group of the C6 carbon of the galactose is protected. (Step C) The hydroxyl groups bonded to the C2, C3 and C4 carbons of the galactose are protected. (Step D) The protecting group of the C6 carbon previously protected is deprotected. (Step E) The hydroxyl group bonded to the C6 carbon is substituted with a group (for example, an alkylsulfonyloxy group or arylsulfonyloxy group) which can be converted to a carbonylthio group. (Step F) The C6 carbon is converted into a carbonylthio group. (Step G) The 2-propenyl group bonded to the C1 carbon is converted into a diol. (Step H) Both of the hydroxyl groups or only the hydroxyl group at the 1-position of the diol thus obtained are/is esterified with a desired higher fatty acid. (Step I) The carbonylthio group at the C6 carbon is converted into a sulfonate salt. (Step J) The protecting groups of C2, C3 and C4 carbons of the sulfonate salt obtained are deprotected. As a result, a salt of a sulfofucosylacylglycerol derivative of the present invention can be produced. The salt thus obtained is subjected to titration with an acid such as hydrochloric acid to give the sulfofucosylacylglycerol derivative of the present invention.
The aforementioned Steps A-J will be further explained in detail.
In Step A, the 2-propenylation is carried out by reacting the galactose with allyl alcohol in the presence of a strong acid, such as trifluoromethanesulfonic acid, usually at room temperature to 100xc2x0 C., preferably from 80 to 90xc2x0 C., for a half day to two days. However, the reaction time varies depending upon the reaction conditions.
In Step B, the hydroxyl group bonded to the C6 carbon is protected to obtain the compound to which -OR6 is bonded at the C6 carbon (where R6 represents an alkyl or substituted silyl group).
As the compound capable of protecting the hydroxyl group, a compound can be used which can provide an alkyl group or substituted silyl group as the R6 group.
Examples of the alkyl group represented by R6 preferably include bulky and substituted alkyl groups. The substituents of the bulky and substituted alkyl groups include methyl and phenyl groups. The specific examples of the substituted alkyl group include t-butyl and trityl groups.
When the group represented by R6 represents a substituted silyl group, examples of substituents of the substituted silyl group include lower alkyl groups, preferably alkyl groups having 1-4 carbon atoms (for example, methyl, ethyl, isopropyl and t-butyl groups); and aryl groups, preferably aryl groups having 6 carbon atoms (for example, a phenyl group). The substituted silyl group represented by R6 preferably includes tri-substituted silyl groups, more preferably, a t-butyldiphenylsilyl group.
When the compound 3, where R6 represents an alkyl group, is to be obtained, the protection of the hydroxyl group in Step B can be carried out by adding a compound represented by R6-X (where R6 represents the alkyl group defined above, and X represents a halogen atom such as chlorine atom) to a solution of the compound 2 dissolved in an organic solvent, such as anhydrous pyridine, and reacting the solution mixture at room temperature in the presence of a catalyst such as p-dimethylaminopyridine (DMAP). As the compound R6-X, trityl chloride is preferably used in view of manufacturability and reactivity.
When the compound 3, where R6 represents a substituted silyl group, is to be obtained, t-butyldiphenylsilyl chloride, for example, is used as the compound R6-X, and the reaction is carried out usually in the presence of a catalyst, such as imidazole, at room temperature for a half day to two days. Note that the reaction time varies depending upon the reaction conditions.
In Step C, the hydroxyl groups bonded to the C2, C3 and C4 carbons are protected and converted into xe2x80x94OR1, xe2x80x94OR2 and xe2x80x94OR3, respectively, where R1 to R3 independently represent an alkyl or substituted silyl group. The protection of these hydroxyl groups can be carried out by activating, with sodium hydride, the hydroxyl groups bonded to the C2, C3 and C4 carbons of the compound 3 dissolved in an organic solvent, such as N,N-dimethylformamide (DMF), and reacting with the compound capable of protecting these hydroxyl groups at room temperature.
As the compound capable of protecting the hydroxyl groups, benzyl bromide, p-methoxybenzyl bromide, t-butyldimethylsilyl chloride or triethylsilyl chloride may be used. Benzyl bromide is preferably used as the protecting group in view of stability of the protecting group in the case where the acyl residue(s) represented by R101 and/or R102 are/is saturated one(s). The reaction using the compound capable of protecting the hydroxyl groups can be carried out under a suitable reaction condition for each of the protecting groups.
The deprotection of the protecting group bonded to the C6 carbon in Step D may be carried out by reacting a solution of the compound 4 dissolved in an organic solvent, such as methanol, in the presence of a catalyst, such as p-toluenesulfonic acid, generally for 12 hours to one day at room temperature. The reaction time varies depending upon the reaction conditions.
In Step E, R4, that is, an alkylsulfonyl or arylsulfonyl group is bonded to the hydroxyl group at the C6 carbon of the compound 5, so that the hydroxyl group is converted into xe2x80x94OR4 to give the compound 6.
The reaction to give the xe2x80x94OR4 group is performed by adding a compound having the alkylsulfonyl group or a compound having the arylsulfonyl group to a solution of the compound 5 dissolved in an organic solvent, and reacting them. The alkyl group of the compound having the alkylsulfonyl group preferably includes unsubstituted alkyl groups, more preferably, lower alkyl groups, much more preferably, alkyl groups having 1-2 carbon atoms (methyl and ethyl groups). The compound having an alkylsulfonyl group can be represented by R4xe2x80x2-x (where R4xe2x80x2 represents an alkylsulfonyl group, and X represents a halogen atom). Specific examples include methanesulfonyl chloride and ethanesulfonyl chloride.
On the other hand, the aryl group of the compound having the arylsulfonyl group may include unsubstituted and substituted aryl groups, preferably aryl groups having 6 carbon atoms (e.g., a phenyl group). In the case of the substituted aryl group, examples of the substituent thereof include p-methyl and p-methoxy groups. Examples of the compound having an arylsulfonyl group include compounds represented by R4xe2x80x3-X (where R4xe2x80x3 represents an arylsulfonyl group, and X represents a halogen atom). Specific examples include p-toluenesulfonyl chloride, p-methoxybenzenesulfonyl chloride and benzenesulfonyl chloride.
Of the compounds having an alkylsulfonyl or arylsulfonyl group, a compound having a p-toluenesulfonyl group (tosyl group) is preferably used from the viewpoint of reaction facility.
In the reaction of Step E, as an organic solvent, for example, pyridine or dichloromethane may be used.
The reaction mentioned above may be performed, as the case may be, in the presence of a catalyst, such as DMAP, at room temperature for 2 hours to one day. The reaction time varies depending upon the reaction conditions.
In Step F, the sulfonyloxy group (xe2x80x94OR4) of the compound 6 is replaced with a carbonylthio group represented by xe2x80x94SC (xe2x95x90O)R5, where R5 represents a hydrogen atom, an alkyl or aryl group.
In the reaction, a compound capable of substituting the alkylsulfonyloxy or arylsulfonyloxy group of the compound 6 with the carbonylthio group, is allowed to react in an organic solvent to give a compound 7. Hereinafter, this compound will be referred to as xe2x80x9cO-substitutedxe2x86x92S-substituted compoundxe2x80x9d.
Examples of the O-substitutedxe2x86x92S-substituted compound include alkali metal salts and alkali earth metal salts of a thiocarboxylic acid. Examples of the thiocarboxylic acid include thioformic acid, lower thiocarboxylic acids, preferably aliphatic thiocarboxylic acids each having 1-2 carbon atoms in its aliphatic hydrocarbon moiety (for example, thioacetic acid or thiopropionic acid), and aromatic thiocarboxylic acids each having 6-10 carbon atoms in its aromatic hydrocarbon moiety (for example, thiobenzoic acid).
The alkali metal that forms a salt with the thiocarboxylic acid includes potassium and sodium. The alkali earth metal includes magnesium and calcium.
Of the above-mentioned O-substitutedxe2x86x92S-substituted compounds, salts of thioacetic acid may be preferably used since a reaction can proceed stably and the sulfur atom can be easily oxidized in a later step.
Examples of an organic solvent used in the reaction include hexamethylphosphoramide, N,N-dimethylformamide and dimethylsulfoxide.
The aforementioned reaction may be performed usually at room temperature to around 100xc2x0 C. while stirring for one hour to one day. Note that the reaction time varies depending upon the reaction conditions.
The dihydroxylation of Step G may be performed by adding an oxidizing agent, such as osmium tetraoxide, to a solution of the compound 7 dissolved in a solvent mixture, such as a mixture of t-butanol and water, and then reacting the resultant mixture in the presence of a re-oxidizing agent, such as trimethylamine N-oxide, at room temperature for one hour to one day. Note that the reaction time varies depending upon the reaction conditions.
By the esterification of Step H, a sulfofucosylacylglycerol derivative having a desired higher fatty acid bonded, through an ester-bond, to its glycerol moiety can be obtained. This reaction can be carried out by adding a fatty acid corresponding to a final product to a solution of the compound 8 dissolved in a suitable organic solvent, such as dichloromethane, and then reacting the resultant mixture, if necessary, in the presence of a suitable catalyst, such as ethyldimethylaminopropylcarbodiimide (EDCI)-DMAP.
In the reaction of Step H, as the fatty acid to be added, use may be made of a higher fatty acid whose acyl group is that represented by R101 of General Formula (1).
In the reaction of Step H, the compound 9 is obtained in the form of a mixture of a diacylester and a monoacylester. The diacylester herein is represented by Formula (1) of the present invention where each of R101 and R102 is an acyl residue of the higher fatty acid added. The monoacylester herein has the acyl residue of the higher fatty acid added, as the R101 only. Two or more higher fatty acids may be added, if desired, in the reaction of Step H. In this case, the resultant mixture contains diacylesters represented by General Formula (1) where R101 and R102 are the same or different acyl residues, and monoesters having different acyl residues as R101.
If necessary, the mixture of the monoesters and diesters can be isolated from each other by, for example, chromatography, and subjected to the next reaction Step I.
Furthermore, if desired, by reacting a monoester obtained in Step H with a fatty acid having a different acyl residue from the acyl residue (R101) of the monoester, it is possible to obtain a diester where R102 and R101 are different acyl residues. This additional esterification step may be performed under the same conditions as those of Step H except that a different fatty acid is used.
In Step I, the conversion into a sulfonate salt can be carried out by adding an oxidizing agent, for example, OXONE (2KHSO5, KHSO4 and K2SO4) into a solution of the compound 9 dissolved in an organic solvent, which is buffered with acetic acid and potassium acetate, and then allowing the resultant mixture to react at room temperature for 12-24 hours. Note that the reaction time varies depending upon the reaction conditions.
The deprotection of the protecting groups bonded to carbons at the C2 to C4 carbons in Step J can be carried out by a method suitable for a protecting group to be used and an acyl residue of the bonded higher fatty acid. For example, when the protecting group is a benzyl group and each of R101 and R102 is an acyl residue of a saturated higher fatty acid, the deprotection can be conducted by reacting a solution of a compound 10 dissolved in an organic solvent, such as ethanol, in the presence of a catalyst, such as palladium-activated carbon, under a hydrogen gas atmosphere at room temperature. Furthermore, when at least one of the acyl residues of the higher fatty acids represented by R101 and R102 is an acyl residue of an unsaturated higher fatty acid, a deprotection method suitable for a protecting group used and capable of retaining the double bond of the unsaturated fatty acid may be employed. For example, when the protecting group is a silyl group, the deprotection can be conducted by use of an acid catalyst (e.g., trifluoroacetic acid).
Note that the galactose of a starting material usually takes xcex1- and xcex2-anomer configurations in a solution. Therefore, the product in each step results in a mixture of xcex1- and xcex2-anomers. The mixture may be separated into xcex1- and xcex2-anomers by chromatography. Furthermore, xcex1-anomer may be separated by carrying out crystallization after Step A.
Now, we will explain the medicaments of the present invention containing at least one compound selected from the group consisting of sulfofucosylacylglycerol derivatives of the present invention and pharmaceutically acceptable salts thereof, as an active ingredient.
The sulfofucosylacylglycerol derivative serving as an active ingredient for the medicaments of the present invention may be an isomer in which the fucosyl moiety is bonded to glyceridyl moiety with an xcex1- or xcex2-configuration. The derivative may be an isomer regarding the asymmetric carbon at the C2 carbon of the glyceridyl moiety. The medicaments of the present invention may include one of these isomers alone or in combination of two or more isomers as long as they do not adversely affect the activity.
In the present invention, the medicinal use includes a DNA polymerase inhibitor and an anticancer agent.
Examples of the pharmaceutically acceptable salts employed in the medicament of the present invention include, but not limited to, a salt of a monovalent cation such as a sodium or potassium ion.
Hereinafter, the compounds of the group consisting of sulfofucosylacylglycerol derivatives and pharmaceutically acceptable salts thereof are sometimes referred to as xe2x80x9cmedicinally active substance of the present inventionxe2x80x9d.
The medicinally active substance of the present invention can be orally or parenterally administered. Medicinally active substance of the present invention can be combined with, for example, a pharmaceutically acceptable excipient or diluent depending on an administration route thereby to form a medicinal formulation.
The forms of the agent suitable for oral administration include, solid-, semi-solid, liquid- and gas-states. Specific examples include, but not limited to, tablet, capsule, powder, granule, solution, suspension, syrup and elixir agents.
In order to formulate the medicinally active substance of the present invention into tablets, capsules, powders, granules, solutions or suspensions, the substance is mixed with a binder, a disintegrating agent and/or a lubricant, and, if necessary, the resultant is mixed with a diluent, a buffer, a wetting agent, a preservative and/or a flavor, by a known method. Examples of the binder include crystalline cellulose, cellulose derivatives, cornstarch and gelatin. Examples of the disintegrating agent include cornstarch, potato starch and sodium carboxymethylcellulose. Examples of the lubricant include talc and magnesium stearate. Furthermore, additives such as lactose and mannitol may also be used as long as they are used conventionally.
Moreover, the medicinally active substance of the present invention may be administered in the form of aerosol or inhalant, which is prepared by charging the active substance of liquid- or fine powder-form, together with a gaseous or liquid spraying agent, and, if necessary, a known auxiliary agent such as a wetting agent, into a non-pressurized container such as an aerosol container or a nebulizer. As the spraying agent, a pressurized gas, for example, dichlorofluoromethane, propane or nitrogen may be used.
For parenteral administration, the medicinally active agent of the present invention can be injected by, for example, rectal administration or injection.
For rectal administration, a suppository may be used. The suppository may be prepared by mixing the medicinally active substance of the present invention with an excipient that can be melted at body temperature but is solid at room temperature, such as cacao butter, carbon wax or polyethylene glycol, and molding the resultant material, by a known method.
For the administration by injection, the medicinally active agent of the present invention can be injected hypodermically, intracutaneously, intravenously or intramuscularly. An injection preparation may be formulated by dissolving, suspending or emulsifying the medicinally active substance of the invention into an aqueous or non-aqueous solvent such as a vegetable oil, a synthetic glyceride with a fatty acid, an ester of a higher fatty acid or propylene glycol by a known method. If desired, a conventional additive such as a solubilizing agent, an osmoregulating agent, an emulsifier, a stabilizer or a preservative, may be added to the preparation.
For formulating the medicinally active substance of the invention into solutions, suspensions, syrups or elixirs, a pharmaceutically acceptable solvent such as sterilized water for injection or normalized physiological saline solution may be used.
The medicinally active substance of the invention may be used together with a pharmaceutically acceptable compound having another activity, to prepare a medicinal preparation.
The dose of the medicinally active substance of the present invention may be appropriately set or adjusted in accordance with an administration form, an administration route, a degree or stage of a target disease, and the like. For example, in the case of oral administration, a dose of the medicinally active substance may be set at 1-10 mg/kg body weight/day. In the case of administration by injection, a dose of the medicinally active substance may be set at 1-5 mg/kg body weight/day. In the case of rectal administration, a dose of the medicinally active substance may be set at 1-5 mg/kg body weight/day. However, the dose is not limited to these.
When the medicinally active substance of the present invention is used as an anticancer agent, examples of cancers to be treated include those having features of malignant tumors such as solid tumors including adenocarcinoma, epithelioma, sarcoma, glioma, melanoma and lymphoma, and a fluid cancer such as leukemia.